Persistent infection with hepatitis C virus is a global public health problem that renders health complications ranging from mild liver dysfunction to hepatocellular carcinoma and end-stage liver disease. The virus-host interactions and molecular mechanisms that support persistent HCV replication and chronic infection are not understood, and we hypothesize that persistence is supported in part through viral control of the innate intracellular immune response to infection. Research conducted under this proposal will focus on understanding the molecular processes by which HCV controls the function and antiviral actions of the host cell interferon regulatory 3 (IRF-3), a critical antiviral effector that signals the host response to infection. Our studies have shown that IRF-3 can render control of HCV RNA replication through the antiviral actions of IRF-3 target genes, and that HCV prevents this response through the actions of the viral NS3/4A protease to block virus-induced IRF-3 phosphorylation and activation. We have identified the NS3/4A-IRF-3 interface as a new therapeutic target in which IRF-3 function can be restored through inhibition of NS3/4A protease activity. We have therefore incorporated molecular-genetic, biochemical and pharmacologic approaches to investigate processes and outcomes of NS3/4A regulation of IRF-3. Studies within our Specific Aims will: 1) Define the processes by which NS3/4A controls IRF-3 phosphorylation, activation and function, 2) Define the contribution of IRF-3 action toward the outcome of novel HCV protease inhibitor therapy, 3) Identify host IRF-3 target genes that regulate HCV RNA replication, and 4) Evaluate the sequence and IRF-3 regulatory function of the NS3/4A coding region isolated from chronic HCV patients harboring various viral genotypes. This work will define the role of the IRF-3-NS3/4A interface and the host IRF-3 pathway in controlling HCV RNA replication and determining the outcome of HCV infection. A unique feature of this proposal is the incorporation of studies to evaluate the contribution of the IRF-3 and host antiviral response toward the overall efficiency of novel protease inhibitor therapy for the treatment of HCV infection. Our studies will define the spectrum of IRF-3 target genes in human hepatocytic cells whose products impart control of HCV RNA replication during therapeutic restoration of the host response to infection.